A considerable amount of traumatic brain injury (TBI) research has focused on the pathologic significance of mechanical and chemical insults (e.g. disturbed calcium homeostasis) to the axons. Yet the precise biochemical mechanisms of axonal injury remain unclear. White matter loss and demyelination have been insufficiently studied in TBI, and we have little knowledge of the biochemical mechanisms of myelin damage. In the past, our laboratories and others have documented over-expression and activation of various proteases (e.g. calpains, caspases, and matrix metalloproteases) following brain injury. In this proposed research, we hypothesize that a subset of structural proteins in axons and myelin are differentially vulnerable to proteolysis after TBI. Inevitably, such proteolysis would result in degradation that significantly compromises the structural and functional integrity of axons and myelin sheaths leading to long-lasting axonal damage and demyelination. The proposed research represents the first systematic examination of the mechanisms of proteolvtic degradation of structural proteins in both axons and mvelin. Our preliminary data have already identified several axonally-enriched proteins that are vulnerable to proteolysis (neurofilament- H, and -L, microtubule-associated protein tau, axolemma-associated amyloid precursor protein [APR] and cytoskeletal proteins alphall- and (betaII-spectrin). Importantly, we have also identified at least two myelin proteins that are proteolysed after TBI (myelin basic protein [MBP] and myelin oligodendrocyte specific protein [MOSP]). Unique in vivo cleavage sites in proteolysis-prone protein substrates will be identified with state-of-the- art proteomic techniques. This knowledge will then allow us to generate novel "fragment-specific" antibodies, which will be used to immunohistochemically examine the precise subcellular distribution of these proteolytic products. The same antibody tools will be used to configure enzyme-linked immunoassays (ELISAs) to quantify these axonal and myelin proteolytic products in both brain tissue and cerebrospinal fluid (CSF). The latter method, if successful, will provide a novel and minimally-invasive way of quantifying TBI-associated axonal and myelin damage. These advances in understanding the proteolvtic mechanisms underlying axonal and myelin pathology in TBI will greatly accelerate development of therapies preserving the structural and functional integrity of axons and myelin.